A new approach to erase fear memory in humans: Vicarious
extinction training interfering with reconsolidation prevents the recovery
of learned fear.
Cathelijn Tjaden (10893520) September 2016
Master Thesis Research Master Psychology
Supervisors:
Dr. Armita Golkar (Karolinska Institutet)
Abstract
Recent research on fear learning demonstrated that disruption of the reconsolidation process using behavioral extinction training seems to be a promising new approach for treating anxiety disorders. However, mixed replication results make it difficult to draw firm conclusions and highlight the need to establish and strengthen the effect. Therefore, the present experiment investigated whether vicarious extinction, an alternative extinction approach that has been shown to out-perform standard extinction training, performed within the reconsolidation time window could prevent the recovery of fear. In a three-day Pavlovian fear-conditioning paradigm, participants (n=18) went through conditioning, vicarious extinction, and reinstatement testing, with one of two reinforced conditioned stimuli (CS) being reminded prior to vicarious extinction. In line with the hypotheses, the results showed that vicarious extinction training conducted after the presentation of a single unreinforced reminder trial prevented the recovery of fear responses to the reminded CS, without affecting responses to the non-reminded CS. Importantly, the study extends previous work by a) using fear-relevant stimuli that are more common objects of fears in pathological fear in anxiety disorders; b) using fear potentiated startle (FPS) as a measurement of fear, that is thought to reflect the affective component of fear; and c) demonstrating a context-independent effect of the vicarious extinction procedure. Hence, the present study provides a new, non-invasive and potentially powerful behavioral technique to disrupt reconsolidation and prevent the recovery of fear.
Keywords: reconsolidation, reactivation, vicarious extinction, observational learning, fear learning, fear-relevant, FPS.
TABLE OF CONTENT
1. Introduction 4
1.1 Reconsolidation 4
1.2 Vicarious extinction 6
1.3 Present study 6
2. Material and Methods 7
2.1 Participants 7
2.2. Apparatus and materials 8
2.2.1 Materials 8
2.2.2 Subjective Assessments 9
2.2.3 Fear Potentiated Startle 9
2.3 Experimental Procedure 10 2.4 Statistical analyses 12 3. Results 13 3.1 Preliminary Analyses 13 3.2 Acquisition 14 3.3 Vicarious extinction 14 3.4 Reinstatement/Re-extinction 14 3.5 Empathy 16
4. Discussion Fout! Bladwijzer niet gedefinieerd.
Reference List 21
Appendix 1: Description and Trial-by –trial graph of the whole sample. 27
1. Introduction
The ability to learn and remember potential dangers in the environment is fundamental for survival. It enables the individual to rapidly form associations between threatening events and preceding cues (e.g. sounds, smells). Hereby, associative learning is an adaptive mechanism that motivates the individual to prepare defense systems before the manifestation of the threat (Frijda, 1986). The most frequently used experimental model to study associative fear learning and memory is Pavlovian fear conditioning, which involves learning the relationships between aversive events (unconditioned stimuli, US) and the environmental stimuli (conditioned stimuli, CS) that predict such events (Bouton, 2002). Whereas learning about predictors of danger is critical for survival, learning to regulate past fears when they no longer serve as relevant cues is just as fundamental for adaptive behavior. Indeed, persistent and excessive fear responses that are no longer proportional to the actual threat of an event lie at the root of pathological fear and anxiety (Mineka & Zinbarg, 2006).
The most frequently used treatment for pathological fear and anxiety is Cognitive Behavioral Therapy (CBT)(Barlow, 2002). A crucial component in CBT is exposure, which consist of repeated exposure to the feared stimulus without its adverse consequence (CS/no-US) (Craske, Treanor, Conway, Zbozinek & Vervliet, 2014). Procedurally, exposure therapy is similar to the fear extinction paradigm used in experimental research, in which the same safety learning procedure (CS/no-US) is applied to weaken the expression of a previously learned fear response. The effectiveness of extinction is typically indicated with the recovery of the conditioned fear response. This can be measured with post extinction phenomena such as reinstatement testing, defined as the recovery of an extinguished conditioned response after re-exposure to the US (Rescorla & Heth, 1975). The recovery of the conditioned fear parallels the clinical phenomenon of relapse after a successfully completed fear-exposure treatment. Indeed, despite the effectiveness of exposure, a substantial proportion of patients experience a relapse (Craske, 1999). Therefore, it is proposed that extinction does not erase fear memories, but instead generates a new inhibitory memory trace (i.e. CS/no-US) that competes with the original fear memory (i.e. CS/US) to determine the behavioral expression of fear (Bouton, 2002). Consequently, recovery of fear can be explained by intact fear memories that reappear. This implies that once a fear association has been established, the fear memory is held to be forever. 1.1 Reconsolidation
Recently however, insights from neuroscience on the process of reconsolidation have challenged this traditional view on fear memory formation. By disrupting reconsolidation, animal studies
have repeatedly been able to change fear memory (Nader, Schafe, & LeDoux, 2000; Sara, 2000; Dudai, 2004). Reconsolidation refers to the process whereby reactivation of a previously consolidated memory renders it malleable, and protein synthesis for re-stabilization is required for the memory to persist (Nader et al., 2000). Reactivation is defined as the use of the conditioned stimulus as a reminder, on the background of the previous consolidated fear memory (Grigor’yan & Markevich, 2015). Once reactivated, memories become labile and are open to modification before they reconsolidate and return to their inactive state. Through reconsolidation, memories can be updated with novel information and manipulations within the window of reconsolidation – the time window after reactivation in which the process of reconsolidation occurs - provide an opportunity to alter unwanted fear memories and facilitate adaption to the environment (Duvarci & Nader, 2004). Evidence for the updating properties of disrupted reconsolidation of fear memory recently progressed from animals to humans. In a series of studies in healthy volunteers the beta-blocker propranolol, administered before or after reactivation of a fear memory (i.e. disrupting reconsolidation), prevented fear memories from recovering (Kindt, Soeter & Vervliet, 2009; Sevenster, Beckers & Kindt, 2012; Soeter & Kindt, 2010). Importantly, it has been proposed that reconsolidation only takes place when reactivation produces an anticipation of threat that generates a mismatch between what is expected and what actually happens (i.e. prediction error) (Sevenster, Beckers & Kindt, 2013).
Altogether, previous work indicates that pharmacologically interfering with the process of reconsolidation can prevent the recovery of consolidated fear memories. However, it is less clear whether a behavioral procedure performed within the reconsolidation-window can similarly prevent the recovery of a fear memory. As pharmacological interventions have significant negative effects on the sufferers perceived ability to self-regulate and self-help (Goodwin et al., 2009), are associated with an increased risk of relapse (Bandelow et al., 2012) and provide only symptomatic treatment (Argyropoulos, Sandford & Nutt, 2000), a behavioral procedure is greatly preferred over pharmacological manipulations. Indeed, if previously acquired fear memories can be updated with safety information using a non-invasive technique interfering with reconsolidation, it might be possible to permanently modify the existing fearful properties of the memory trace and persistently reduce recovery of fear. To date, the most influential behavioral strategy was reported by Schiller and colleagues (2010), suggesting that extinction training after the presentation of a reminder cue of a fear memory can update the fear memory with non-fearful information, herewith preventing the recovery of fear (Schiller, Monfils, Raio, Johnson, LeDoux & Phelps, 2010). That is, participants undergoing extinction training 10 minutes after exposure to a single, non-reinforced reminder trial of a previously acquired conditioned fear
showed decreased recovery of fear in a re-extinction session 24 hours later and up to one year later (Schiller et al., 2010). Some replication studies confirmed that behavioral extinction training conducted after a single reminder exposure could prevent the recovery of fear (Oyarzún et al., 2012; Agren et al., 2012a; Agren, Fumark, Eriksson, Frederikson, 2012b). However, others failed in replicating and found no indication of the effect, suggesting that it is fragile (Soeter & Kindt, 2011; Kindt & Soeter, 2011; Golkar, Bellander, Olsson & Öhman, 2012; Klucken, Kruse, Schweckendiek, Kuepper, Mueller, Hennig & Stark, 2016).
1.2 Vicarious extinction
In order to stabilize the effect of extinction training during reconsolidation, an alternative safety procedure could provide more solid outcomes. Such a promising alternative procedure was recently offered by Golkar and colleagues, who reported that vicarious extinction learning out-performed standard extinction learning in short-term recovery of fear (Golkar, Selbing, Flygare, Ohman & Olsson, 2013). Vicarious extinction learning is defined as a form of modeling in which a previously reinforced behavior is eliminated by withdrawal of consequences applied to a model. It is used as part of exposure treatments of phobias in which the client views the therapist who approaches and interacts with the phobic stimulus before the phobic patient is directly exposed to it (Seligman & Wuyek, 2005). The principle underlying such treatment can be explained by social learning, emphasizing that socially transmitted information is a less risky route to achieve information about potentially dangerous or harmful situations (Olsson & Phelps, 2007). Capitalizing on the same principle, Golkar and colleagues (2013) reported that – using a standard fear conditioning protocol –safety information acquired through vicarious extinction learning prevented the recovery of short-term fear memory more effectively than what was achieved with standard extinction training.
1.3 Present study
By combining the efficacy of disrupting reconsolidation to update old fear memories in humans (Kindt et al., 2009) and the strong effect of vicarious extinction learning in preventing the recovery of short-term fear memory (Golkar et al., 2013), the present study will examine whether vicarious extinction training conducted after the presentation of a single reminder trial may offer a strong, behavioral and non-invasive alternative to change consolidated fear memories. Hypothetically, if the reminder trial of a previously fear conditioned cue triggers a specific threat expectation, the non-occurrence of the anticipated aversive event , as seen by the calm reaction of the model, could be experienced as a mismatch between what was expected and what actually happened (i.e., prediction error). Hereby, the original fear association undergoes reconsolidation,
making the fear memory increasingly sensitive to disruption and prone to updates by the vicarious extinction learning. Notably, the efficacy of the vicarious extinction learning should be restricted to the reminded stimuli and should not impact the non-reminded stimuli. In line with previous research interfering reconsolidation using a behavioral approach (i.e. Schiller et al., 2010, 2013; Kindt & Soeter, 2011; Golkar et al., 2013), the present study used a subsequent three-day Pavlovian fear-conditioning paradigm, involving fear conditioning (Day 1), reminder - vicarious extinction learning (Day 2) and test of fear recovery (Day 3). Additionally, the present study will use fear potentiated startle (FPS) as a measurement of fear as this has been reported to be a more specific index of emotional fear learning than the frequently used Skin Conductance Response (SCR) (Sevenster, Becker & Kindt, 2014), and fear-relevant stimuli as they thought to be more representative of the severe pathological fear in anxiety disorders (Mineka & Öhman 2002). Notably, a behavioral intervention performed after the presentation of a reminder trial has never been reported to be successful using these parameters. It was hypothesized that vicarious extinction training following a single, unreinforced reminder trial of a fear conditioned cue would result in less reinstatement of fear, compared to vicarious extinction training of a non-reminded fear conditioned cue. Secondly, as empathy has been shown to be a modulating factor in vicarious fear learning (Olsson, McMahon, Papenberg, Zaki, Bolger & Ochsner, 2016), the present study will exploratory investigate if this also applies to vicarious safety learning by assessing whether trait empathy is related to the efficacy of vicarious extinction learning. In other words, if individuals high in trait empathy benefit more from the vicarious extinction procedure by assessing whether high scores on trait empathy are related to lower fear responses during vicarious extinction learning and reinstatement-testing.
2. Material and Methods 2.1 Participants
Thirty-nine undergraduate students from the University of Amsterdam (UvA) completed all three days of the study. Participants were recruited by online advertisement linked to the UvA. Two participants were excluded for statistical analysis after medical screening and their data wasone participant was excluded due to a technical failure to administer the reinstatement shocks at Day 3. Moreover, one participant was excluded after incorrect reporting of the CS-US contingency at Day 1 and three participants were excluded after reporting the model receiving shocks at Day 2. In line with previous reasoning (see Kindt & Soeter, 2011, Schiller et al., 2010, 2013), fear recovery cannot be assessed accurately when fear responses are not successfully acquired. Consequently, fourteen participants were eliminated because they failed to condition fear to
either one or to both the CS+s, as assessed by differential Fear Potentiated Startle (FPS). That is, successful acquisition was defined as a larger CS+/CS- startle differentiation on the last two trials of acquisition (post-learning) when compared to the first two trials of acquisition (pre-learning) for the reminded conditioned stimulus (CS+r ) vs. the neutral stimulus (CS-) and the non-reminded conditioned stimulus (CS+nr) vs. the neutral stimulus (CS-) separately. See appendix 1 for the description and the trial-by-trial figure of the sample before exclusion based on acquisition (n=32). The final sample consisted of 18 healthy undergraduate students (4 males; age M = 20.6 years, SD = 1.4 years, range = 19-23 years). The ethical committee of the University of Amsterdam approved the study and informed consent was obtained from all participants. Participants were rewarded by either partial course credits or small payment (€40, -).
2.2. Apparatus and materials 2.2.1 Materials
Two fear-relevant stimuli of different categories (spider: IAPS 1200 and gun: IAPS 6210) served as the conditioned stimuli (CS) (Lang, Bradley & Cuthbert, 2005; Öhman & Mineka, 2001) and a fear-irrelevant image of a blue mug served as the neutral CS- (IAPS 7009; see Kindt & Soeter, 2011 for a similar approach), see Figure 1. The slides were 200 mm high and 270 mm wide, and were presented in the middle of the screen of a 19-inch computer screen over a black background. Screen resolution was 800 × 600 pixels and the refresh rate was set to 60 Hz. The startle probe (40 ms; 104 dB) was presented 7s after the CS onset and during noise alone trials (NA trials). The probe was binaurally delivered through headphones (Model MD-4600; Compact Disc Digital Audio, Monacor) and was, for the CS+r/CS+nr, followed by the unconditioned stimulus (US) 500ms later (i.e. at 7.5 s), see Figure 2.
The US consisted of an electric stimulus with duration of 2 ms, administrated to the left wrist. The electric stimulus was delivered via a pair of Ag electrodes of 20 mm × 25 mm and controlled by a Digitimer DS7A (Hertfordshire, UK). A conductive gel (Signa, Parker) was applied between the electrodes and the skin. The experiment was programmed in Presentation 13.1 (Neurobehavioral Systems, www.neurobs.com). For the vicarious extinction training, a video-clip of 24 min was created using Adobe Premiere Pro CS5.5. The clip contained a learning model that was seated in front of a black screen (see Figure 2). The model was attached to similar equipment and reacted calmly to unreinforced presentations of all the three CS’s that were shown on his screen (CS+r, CS+nr and CS-) (Golkar et al., 2013).
a) b) c)
Figure 1. a) Stimuli that served as the two CS+s. b) The neutral stimulus CS- (never paired with a shock). c) The model on the video that the participants watched, who was performing the vicarious extinction training.
2.2.2 Subjective Assessments
Trait anxiety was assessed with the self-rating questionnaire Trait Anxiety Inventory (STAI-T) (Spielberger, Gorsuch & Lusthene, 1970) and the degree of spider fear was rated by the FSQ (Szymanski & O’Donohue, 1995). Moreover, trait empathy was measured with the scores on the Interpersonal Reactivity Index (IRI; Davis, 1980). For an elaborate description and psychometric characteristics of these questionnaires, see Appendix 2. In addition, the US was evaluated after Day 1 and Day 3 by a Likert-scale assessing unpleasantness, intensity and effort to resist; and the learning model on Day 2 was evaluated on Likert-scales assessing trustworthiness, self-identification, attractiveness and empathic concern.
2.2.3 Fear Potentiated Startle
The conditioned response (CR) of fear was measured as potentiation of the eyeblink startle reflex to a loud noise by electromyography (EMG) of the right orbicularis oculi muscle. A potentiated contraction (i.e. potentiation) of the startle blink response is observed when the organism is in a fearful state (Weike, Schupp & Hamm, 2007). Neurally, this reflects the influence of direct and indirect connections from the amygdala to the primary startle-reflex pathway in the brainstem (Davis & Whalen, 2001). In order to capture the EMG signal, two 7 mm Ag/AgCl electrodes were filled with electrolyte gel and positioned approximately 1 cm under the pupil and 1 cm below the lateral canthus, a third ground reference was placed on the forehead. The raw EMG signal was measured using a bundled pair of electrode wires connected to a front-end amplifier with an input resistance of 10 MΩ (omega) and a bandwidth of DC-1500 Hz. To remove unwanted interference, a notch filter was set at 50 Hz. Integration was handled by a true-RMS converter (contour follower) with a time constant of 25 ms. The integrated EMG signal was sampled at 1000 Hz. Startle eye-blink magnitude (microvolts) was measured as the amplitude
from onset to peak (over the period of 50 – 200 ms following probe onset) and normalized for each participant using t-standardization (Blumenthal et al., 2005) deriving a distribution with overall mean 50 and standard deviation 10.
2.3 Experimental Procedure
The procedure involved a Pavlovian differential fear conditioning procedure across three subsequent days, conducted 24h apart (see Figure 1). During each session, participants sat behind a table with a computer screen at a distance of about 50 cm in a sound-attenuated room. Assignment of the spider and gun images as the reminded conditioned stimulus was counterbalanced across participants, with the restriction that the two conditions were matched on the scores on the Fear of Spiders Questionnaire (FSQ; administered before the start of the experiment) as close as possible1. All three days began with a habituation phase consisting of ten
startle probes. Several phases were completed across the three days: acquisition (Day 1); reminder trial and vicarious extinction (Day 2) and reinstatement and re- extinction (Day 3) (see Figure 1), using a within subjects design. Briefly, participants underwent fear conditioning on Day 1 using three different CSs. The two CS+s (CS+r = CS+ reminded and CS+nr = CS+ non-reminded) were paired with the US (80% reinforcement) whereas the neutral third CS (CS−) was never paired with the US. A day later, participants received a single presentation of the CS+r, but not the CS+nr, and underwent vicarious extinction training. At Day 3, participants received 3 un-signaled USs, followed by re-extinction, consisting of CS-presentations without the US.
2.3.1. Day 1: Acquisition.
At the beginning of the session, participants were welcomed and interviewed regarding their health and any medical conditions. Moreover, written informed consent was obtained, possible questions were answered and questionnaires assessing trait anxiety (STAI-T, Spielberger et al, 1970) and trait empathy (IRI, Davis, 1980) were administered. After attachment of the startle electrodes (right-eye) and shock electrode (left wrist), the intensity of the US was adjusted for each participant individually by gradually increasing the level of the aversive electric stimulus until the level was “unpleasant but not painful”. The participants were told that the intensity of the electric stimulus would remain set throughout all three days. Then the participants were informed (both orally and by written instructions on the screen) that two of the presented images would mostly be followed by the electric stimulus, whereas the third image would never be followed by the
1 The counterbalancing did not lead to differences in terms of reported spider fear (FSQ), trait anxiety (STAI-T),
electric stimulus. Participants were instructed to pay close attention to the contingency between the pictures and the US administration, so that they could predict after which image they would receive a shock. Moreover, the importance of remembering this contingency was emphasized, as they were told that there would follow questions about this at the end of the experiment. Finally, the participants were explicitly told to remain focused during the task with their gaze to the screen and that the researcher would keep an eye on them and the experiment through the camera.
Then the experiment started with acquisition in which two fear-relevant stimuli (CS+r, CS+nr) and one neutral stimulus (CS-) were presented 5 times for 8s. The startle probe was presented 7s after CS onset and was followed by the US 500ms later (i.e. at 7.5s) on reinforced trials, see Figure 2. Each CS was presented in a pseudo-randomized order with the criterion that there could be no more than two trials of the same CS in a row. Moreover, the first acquisition trial for both the CS+s was always unreinforced. The images of the two CS+s (CS+r, CS+nr) were partly reinforced (80%) to prevent that the reminder trial on day 2 would result in extinction learning. In addition, 5 startle probes were presented alone (NA trial). Furthermore, after each trial an inter-trial interval (ITI trial) was inserted, varying between 15s, 20s, and 25s, with a mean of 20s. At the end of Day 1, the participants were asked to report the CS-US contingency and to evaluate the US. Furthermore, participants were instructed to remember what they had learned during the acquisition phase of the experiment to enhance retention of the CS-US contingency on the following days (Norrholm et al., 2006) and to prevent that participants would incorrectly expect a different contingency for the two subsequent days. Finally, participants were asked not to talk with other students about the experiment. To accomplish consolidation of the fear memory (Dudai, 2004), there was a break of at least 24 hours after the acquisition phase.
2.3.2. Day 2: Reminder Trial and Vicarious Extinction.
After electrode attachment, participants were told that the same three images would be presented on the screen and they were instructed to remind what they learned the previous day. In order to remind the acquisition memory, a single unreinforced CS+ (CS+r) was presented for 8s, followed
by a startle probe presented alone (NA). After the presentation of the single reminder trial participants were detached from the shock electrode, the other experimental set-up (startle electrodes and skin conductance) remained attached. Then, a 10 min break was inserted (Schiller et al., 2010) in which participants were offered magazines to read. After the break and prior to vicarious extinction learning, the shock electrode was reattached and participants were instructed that they would watch a movie clip of another person with similar instruments and performing a
similar task. Once again, the importance of staying focused was emphasized. At the end of Day 2, the participants were asked whether they and/or the model received shocks and to evaluate the model.
2.3.3. Day 3: Reinstatement and Re-extinction.
After electrode attachment, participants were instructed that the same images as those of the previous days would be presented. The session started with three un-signaled presentations of the US (intensity was set at the same level as during acquisition), after which participants were given a 10-min break, again reading magazines while detached from the shock-electrode. Re-extinction followed immediately after the break and consisted of 5 non-reinforced presentations of each CS (CS+r, CS+nr, CS-), and 5 NA trials. At the end of Day 3, the participants were asked to report the CS-US contingency and to evaluate the US. Moreover, possible questions were answered and the participants were thanked for their participation.
Figure 2. Throughout all stages, each CS is presented for 8 s; startle probes are presented after 7s and during noise alone trials (NA). During Day 1, CS+s are followed by the US 500ms before their offset (partial reinforcement: 80%).
2.4 Statistical Analyses
Data were analyzed with SPSS version 22.0. Each phase of the experiment (acquisition, vicarious extinction, and reinstatement/re-extinction) was analyzed separately by means of a 3 x 2 analysis of variance (ANOVA) for repeated measures with Stimulus (CS+r, CS+nr, CS-) and Block (early, late responses within each phase) as within-subjects factors. For acquisition and
Day 1
Acquisition 24 h
Day 2
Reminder trial – Vicarious extinction
Day 3
Reinstatement – Re-extinction 24 h
Acquisition Reminder Break Vicarious
extinction Reinstatement Break Re-extinction
5 CS+r 1 CS+r 1 NA 10 min 9 CS+r 3 US 10 min 5 CS+r 5 CS+nr 10 CS+nr 5 CS+nr 5 CS- 5 NA 10 CS- 9 NA 5 CS- 5 NA
reinstatement/re-extinction, early and late Block was defined as the mean of the first two and last two trials respectively. As vicarious extinction contained double the amount of trials, early and late Block was defined as the mean of the first four and last four trials, respectively. Moreover, as in previous work (Norrholm et al., 2006), mean startle magnitude for each trial type (CS+r, CS+nr or CS-) in each block was used to calculate the difference score with the ITI trials using the following formula:
Difference Score = [Mean startle magnitude to startle probe in the presence of one of the three trial types (CS+r, CS+nr or CS-)] – [Mean startle magnitude to startle probe alone (ITI)].
Furthermore, Pearson correlations were performed in order to assess the relation between trait empathy, as assessed by means of scores on the IRI, and mean FPS scores on Day 2 and Day 3. Overall, significance was taken at p < 0.05 and partial η2 was reported as the estimate of the effect
size. Greenhouse-Geisser adjustments of degrees of freedom were used in case of violated sphericity, as measured with Mauchly’s W test. Significant interactions and pre-planned comparisons were followed up with separate two-tailed paired t-tests.
3. Results
3.1 Preliminary Analyses
Assigning the spider or gun image as the CS+r across participants did not lead to differences in age, trait anxiety (STAI-T), reported spider fear (FSQ), selected US intensity (US level), perceived US intensity at Day 1 and perceived US intensity at Day 3, see Table 1.
Table 1
Mean values (SD) of age, trait anxiety, reported spider fear, level of the unconditioned stimulus (mA) and perceived intensity of the US (n=18) in the two groups.
Spider = CS+r (n=11) Gun = CS+r (n=7) t-value (df = 16)
Age 20.90 (1.45) 20.00 (1.16) 1.40, p = .181 STAI Trait 32.18 (7.61) 35.00 (7.62) -0.77, p = .455 FSQ 29.73 (15.65) 31.29 (6.34) -0.25, p = .807 US Level 38.32 (25.31) 25.29 (13.54) 1.25, p = .231 US Intensity Day 1 2.72 (0.79) 3.14 (0.38) -1.30, p = .213 US Intensity Day 3 2.55 (0.52) 2.86 (0.69) -1.09, p = .291
3.2 Acquisition
For the trial-by-trial graph of all stages see Figure 3. At day 1, the 3 (Stimulus: CS+r, CS+nr, CS-) x 2 (Block: early, late) ANOVA showed successful fear learning by a significant increase of the startle fear responses from early (first two trials) to late (last two trials) acquisition, as supported by a significant Stimulus x Block interaction effect2, F
(2,34) = 31.896, p = .000, η2= .652.
Follow-up t-tests confirmed that FPS responses to the CS+r and the CS+nr were significantly higher than to the CS- both during late acquisition, (t(17) = 4.747, p = .000 and t(17) = 4.601, p = .000
respectively), and after acquisition (last trial; t(17) = 3.690, p = .002 and t(17) = 3.738, p = .002,
respectively). Importantly, fear responses to the two CS+s were equally acquired during late acquisition (t(17) = -0.920, p = .371) and after acquisition (last trial:t(17) = -1.197, p = .248); and
equally consolidated over 24 h, as indicated with the absence of a significant change from the last presentation at acquisition to the first presentation at day 2 for both CS+s (all t’s <2).
3.3 Vicarious extinction
During day 2, FPS responses were overall higher at early vicarious extinction than at late vicarious extinction, as indicated with a main effect of Block, F(1,17)= 10.125, p = 0.005, η2=
0.3732. Importantly, successful fear extinction was evidenced by a significant decrease in FPS
responses from late acquisition to late vicarious extinction to the CS+r (t(17) = 3.398, p = .003)
and to the CS+nr, (t(17) = 4.036, p = 0.001). Moreover, planned comparisons revealed that there
were no differences in responses to both CS+s and the responses to the CS- at late vicarious extinction (CS+r vs. CS-: t(17) = 1.886, p = .077 and CS+nr vs. CS-: t(17) = 1.764, p = .096,) and
after vicarious extinction (last trial; t(17) = 1.008, p = .327; t(17) = 1.463, p = .162, respectively),
demonstrating that the fear learned on day 1 extinguished with the vicarious extinction procedure on day 2. Furthermore, the CS+r and the CS+nr extinguished equally, as shown with the absence of significant differences at late vicarious extinction (t(17) = 0.504, p = 0.621), and after vicarious
extinction (last trial; t(17) = -.845, p = .410).
3.4 Reinstatement/Re-extinction
In line with similar previous work of behavioral interference with reconsolidation (i.e. Schiller et al., 2010; 2013), fear responses to the three CSs were analyzed from the end of vicarious extinction (Day 2) to the first block of re-extinction (Day 3). Paired sample t-tests revealed that
2 Sphericity was assumed as the Mauchly’s W test was not significant.
there was a significant increase in FPS only to the CS+nr (: t(17)= 2.45, p = 0.026) but not to the CS+r and the CS- (t(17) = .50, p = 0.624; t(17) = .62, p = 0.545, respectively). In addition, during the first block of re-extinction testing, FPS responses were greater to the CS+nr when compared to the CS+r (t(17) = 2.88, p = .010), and only FPS responses to the CS+nr significantly differed from those to the CS− (CS+nr vs. CS-: t(17) = 2.801, p = .012; CS+r vs. CS- t(17) = .655, p = .52). Moreover, a 3 (Stimulus) x 2 (Block) ANOVA2 for the re-extinction phase revealed a
significant main effect of Stimulus (F(2,34)= 6.056, p = .006, η2 = .263), indicating that the
differences between the CS+nr and the other CSs remained from the first to the last block of re-extinction; and a main effect of Block (F(1,17) = 23.79. p = .000, η2= .58), indicating that the CS
responses decreased equally across blocks. Additionally, after visual inspection of the trial-by-trial data, mean CS responses across the re-extinction stage were compared. This analysis confirmed that only the mean response to the CS+nr differed from those to the CS- (CS+nr vs. CS-: t(17) =
3.435, p = .003 and CS+r vs. CS-: t(17) = 1.298, p = .212) and that the mean response to the CS+r
was significantly lower than the mean response to the CS+nr (t(17) = -3.118, p = .006). Post-hoc
power computation showed a probability of .86 to detect the average difference between the two CS+s.
Figure 3. Trial-by-trial mean fear-potentiated startle (FPS) responses (n=18) elicited during the presentation of each stimulus (CS+r, CS+nr and CS-), and during the noise alone trials (NA) 40 45 50 55 60 65 70
A1 A2 A3 A4 A5 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 R1 R2 R3 R4 R5
Fe ar P ot en ti at ed S ta rt le ( T -sc or e) CS+reminded CS+ non-reminded CS- Reminder
across the three stages: Acquisition (A1-A5), Vicarious Extinction (E1-E10), and Re-extinction (R1-R5). Error bars represent the standard error of the mean (SEM). The Y-axis is scaled to T-scores.
3.5 Empathy
With a Pearson bivariate correlational analysis it was explored whether individual differences in empathy were related to individual differences in the FPS responses during a) the vicarious extinction training on Day 2 and b) the re-extinction stage on Day 3. Table 2 provides a summary of the found correlational relations between the FPS scores (Day 2 and Day 3) and IRI scores (Total Score and scores of the subscales: (Perspective Taking (PT), Fantasy (F), Empathic Concertn (EC) and Personal Distress (PD)). These results indicate that trait empathy is not related to better vicarious extinction learning (Day 2) or to less reinstated fear the day after the procedure (Day 3).
Table 2
Pearson Correlations Between IRI Empathy Scores And Mean FPS Scores (n=18).
4. Discussion
The ability to modify established emotional memories has important implications for the treatment of many mental disorders, including anxiety disorders (Schwabe, Nader, Pruessner, 2014). Animal and human studies have repeatedly been able to change fear memory by disrupting the process of reconsolidation of a fear memory with pharmaceutical manipulations (Nader et al., 2000; Kindt & Soeter, 2011). Recently, it was demonstrated that behavioral extinction training conducted within the reconsolidation window could prevent the recovery of fear in memories (Schiller et al., 2010). However, inconsistent replication attempts (i.e. Soeter & Kindt, 2011; Kindt & Soeter, 2011; Golkar et al., 2012; Klucken et al., 2016) make it difficult to draw firm conclusions and highlight the need to establish and strengthen the effect. Therefore, the present study investigated whether performing vicarious extinction, an alternative extinction approach that has been shown to out-perform standard extinction training (Golkar et al., 2013), within the
IRI Total Score r- value (df = 16) IRI PT r- value (df = 16) IRI F r- value (df = 16) IRI EC r- value (df = 16) IRI PD r- value (df = 16) Mean FPS Day 2 -.144, p =.568 -.236, p = .345 .295, p = .235 -.070, p = .781 -.437, p = .070 Mean FPS Day 3 .284, p = .253 .205, p = .461 -.026, p = .917 .224, p = .372 .334, p = .175
vicarious extinction training conducted after the presentation of a single reminder trial targeted specific fear memories and prevented previously learned fear responses from recovering. Hence, this study provides a new, non-invasive and potentially powerful behavioral technique to disrupt reconsolidation and adds to previous work in at least three ways.
First, the present study is the first in showing that a behavioral manipulation after the presentation of a reminder trial successfully prevents the recovery of fear using fear relevant stimuli. Given that anxiety disorders do not tend to be associated with fear-irrelevant stimuli (e.g., geometric figures), but rather with objects and situations related to survival threats (i.e., spiders, guns) (Mineka & Öhman, 2002), the present study was directed towards updating stronger fear memory with a behavioral procedure. Visual inspection of the present data confirms a pattern that can be expected when using fear relevant stimuli. That is, during re-extinction the non-reminded CS decreased slowly across trials and mean responses to the non-non-reminded CS differed from the mean of the CS- throughout the re-extinction trials. In contrast, the prevented recovery of fear of the reminded CS did not follow this pattern and responses towards the reminded CS+ diminished to a level comparable with the CS-. Additionally, the difference in fear reaction between the reminded and the non-reminded CS+ was not restricted to the first trial, as has been the case in previous reconsolidation studies (Schiller et al., 2010; 2013; Oyarzún et al., 2012; Agren et al., 2012a) but maintained throughout the course of re-extinction, as was measured with the mean of FPS responses to both CS’s. Considering clinical purposes, interfering reconsolidation of a stronger fear memory (e.g. fear relevant stimuli) is an important and promising finding opening up new possibilities for future investigations.
Second, the present study is the first in showing a successful effect of a behavioral manipulation conducted after the presentation of a single reminder trial, using FPS as a measurement of fear learning. This extends previous work indexing fear learning with Skin Conductance Responses (SCR). It has been argued that SCR, although traditionally regarded as a
measure of fear (Prokasy & Kumpfer, 1973), evaluates the declarative knowledge - relations
among the events in the environment on a cognitive level - about the stimulus contingencies, relying among other brain areas on the hippocampal complex (Hamm & Weike, 2005). In contrast, the startle response, an automatic defensive reflex, more closely corresponds to the
affective component of fear learning (Davis, 2006) and reflects an amygdala-initiated response
(Walker & Davis, 2002). Nonetheless, fear is considered to be characterized by both high arousal and negative valence (Lang, 1995). Whereas negative valence is a necessary condition for startle potentiation to occur, SCR responses can be established independent from negative valence and is also observed for non-aversive, but arousing events (e.g., reaction time tasks or positive
pictures) (Hamm & Vaitl, 1996). Therefore, as SCR seems to primarily reflect ‘anticipatory arousal’ (Weike et al., 2007), it has been argued that FPS represents a more specific index of emotional fear learning than SCR (Sevenster et al., 2014). Additionally, the affective component is considered to be more discomforting in clinical anxiety disorders (Steimer, 2002), illustrating the potential clinical value of the current findings.
Third, this study is the first to demonstrate a context-independent effect following a single reminder trial of a previously conditioned fear memory. That is, whereas acquisition and re-extinction learning occurred in the same context (direct CS presentations in the absence of the model), safety learning during vicarious extinction was performed in a separate context (indirect CS presentations in the presence of the model), suggesting that the effects of vicarious extinction learning “survived” the context change. Generally, extinction learning is found to be highly context specific (Bouton, 2002) and overcoming the contextual dependency of exposure-based procedures in Clinical Behavioral Therapy (CBT) represents one of the challenges in sustaining the efficacy of exposure-based protocols (Vervliet, Baeyens, Van den Bergh & Hermans, 2013). Therefore, the finding that the safety information obtained by vicarious extinction learning performed after a reminder trial generalized to a new context may be of particular relevance for understanding and treating the persistence of fear memories in individuals suffering from emotional disorders.
Furthermore, the demonstrated efficacy of the vicarious extinction procedure in the present study suggests that model-based learning may help to optimize exposure treatment. In such treatment, the anxious individual watches the therapist—acting as a learning model— approach and interact with the feared stimulus before the individual is directly exposed to it (Seligman & Wuyek, 2005). Previous studies have shown that vicarious extinction promotes better extinction compared to a standard extinction procedure and effectively blocked the recovery of previously learned fear (Golkar et al., 2013). Importantly, it was demonstrated that the presence of a learning model per se during extinction is not sufficient to explain the blockade of reinstatement following vicarious extinction. Rather, the effect was specifically driven by the model’s experience of safety (Golkar et al., 2013). Moreover, vicarious extinction learning has been shown to be associated with a decreased engagement of the vmPFC-amygdala circuitry (Golkar, Haaker, Selbing & Olsson, 2016), contrary to what is expected from standard extinction learning. It was suggested that the added safety signal obtained by a shared safety experience might reduce the necessity for PFC-mediated inhibition during learning that is typically observed in traditional extinction procedures (Phelps, Delgado, Nearing & LeDoux, 2004; Golkar et al., 2016). Interestingly, the same neural pattern was recently reported following successful extinction
training within the reconsolidation time-window (Schiller, Kanen, LeDoux, Monfils & Phelps, 2013), suggesting that both procedures are associated with neural mechanisms that are distinct from standard extinction only.
Notwithstanding, social safety learning is bound to certain conditions. For example, safety learning transmitted from a racial in-group demonstrator was more potent when compared to a racial out-group demonstrator (Golkar, Castro & Olsson, 2015), demonstrating that social safety learning relies on the transmission of social information and can be enhanced depending on the social relationship between the observer and the demonstrator. Future research should address whether similar boundaries applies to the effects reported in the present study. Additionally, a subsample of the participants in the present study erroneously reported that the model received shocks during the extinction session (n=3), similar to previous studies investigating vicarious extinction (Golkar et al., 2013). As the safety of the learning model is a key aspect of the procedure, they were excluded from the final analysis. Nevertheless, as this subsample did not understand (or did not attend to) the safety message of the model, the vicarious extinction procedure may not be effective in all individuals (Golkar et al., 2013). Previous research investigating vicarious fear learning indicated empathy as a modulating factor in learning about threats by observing others (Olsson et al., 2016). In an attempt to investigate whether empathy is also involved in vicarious safety learning, exploratory correlational analyses were conducted in the present study. These analyses did not reveal a significant influence of empathy. However, due to the limited number of participants (n= 18), exploratory analysis resulting in null findings should be evaluated with caution. Future, well-powered research should be aimed at exploring features that discriminate between participants that benefit and those that don’t benefit from vicarious safety learning.
Although the present findings suggest that vicarious extinction initiated after the presentation of a single reminder cue may provide an efficient procedure to prevent the recovery of fear, there are several limitations to the findings. First, although the power of detecting the crucial difference between the two CS+s during the re-extinction phase was still remarkably high (post-hoc power .86), the final sample size of the present study was limited to 18 participants. Although this is comparable to previous reconsolidation studies reporting successful prevention of fear (Schiller, 2010, 2013), follow-up investigations should replicate the present findings in larger samples. Additionally, the results of the present study do not directly explain the mixed replication results of a standard extinction procedure after a reminder trial because vicarious extinction and standard extinction were not directly contrasted in the design. That is, as described before, previous studies have not been able to show a consistent effect of standard extinction
after the presentation of a reminder trial, suggesting that the effect is fragile. Although the present results provide convincing evidence that vicarious extinction learning offers a strong alternative extinction procedure, future research should contrast the two extinction procedures in order to draw firm conclusions.
Moreover, by directly contrasting the effect of vicarious extinction alone and in combination with the presentation of a reminder trial, the present study underlines the essential role of the reminder trial in the effects of the vicarious extinction procedure. Although this strongly suggests that the reminder trial activated the process of reconsolidation, to date, there is no agreement on how to verify whether reconsolidation was targeted (Pedreira, Pérez-Cuesta & Maldonado, 2004). Indeed, past research demonstrated that mere presenting the reminder trial of the previously learned fear memory is not sufficient to induce its labialization and subsequent reconsolidation (Sevenster, Beckers & Kindt, 2012) and fear expression during the presentation of the reminder trial is not informative on whether the memory trace enters the desired labile phase (Sevenster, Beckers & Kindt, 2013). Consequently, from the present design it cannot be concluded that the reminder trial led to reactivation whereby the reconsolidation window has been targeted. The ongoing debate and the inconsistent findings regarding behavioral manipulations conducted after a reminder trial ask for further critical testing to unravel the optimal and boundary conditions for targeting the process of reconsolidation in order to update fear memory through behavioral manipulations.
Furthermore, on a more general level, there is a need for experimental procedures that discriminate between the processes of reconsolidation and extinction, as they lead to different long-term outcomes and therefore might explain the different results (Vervliet et al., 2013). Indeed, some cognitive behavioral interventions (for example the computer game tetris (James et al., 2015)) may already capitalize on disrupting reconsolidation of the underlying fear memories Merlo, Milton, Goozée, Theobald & Everitt, 2014). However, the issue of whether specific interventions either weaken the original fear memory or whether a new extinction memory is formed is currently unresolved (Kindt & Soeter, 2011). For example, reminding the fear memory triggers reconsolidation, but at the same time all extinction learning starts with reminders of the fear memory (Vervliet et al., 2013). In addition, in clinical practice most clients might already remind their fear memories to a certain degree whenever they enter treatment sessions. The question then is what type, degree, or frequency of reminding opens the reconsolidation window and thereby provides the opportunity to update the underlying memories (Kindt, 2014; Vervliet et al., 2013). Some boundary conditions have already been identified such as the age of the fear memory (Wang, de Oliveira Alvares & Nader, 2009), the frequency of the presentations of
memory reminder trials (Wichert, Wolf & Schwabe, 2013), the context in which the presentation of the reminder trials takes place (Hupbach, Gomez & Nadel, 2009) and the reminder structure of the fear memory (Forcato, Argibay, Pedreira & Maldonado, 2009). Although some of these boundaries were not found in replication attempts (Nader & Hardt, 2009), understanding the conditions under which fear memories undergo reconsolidation is crucial in applying reconsolidation as a treatment strategy for anxiety and emotional disorders.
In sum, the present study provides a new, non-invasive and potentially powerful behavioral technique to disrupt reconsolidation and prevent the recovery of fear. Using a three-day Pavlovian fear-conditioning paradigm, the present study revealed that vicarious extinction conducted after the presentation of a reminder cue of a previous learned fear memory could prevent the recovery of fear responses. Moreover, vicarious extinction training only affected the fear memory of the reminded conditioned stimulus, while leaving FPS responses of the other, non-reminded conditioned stimulus intact. Importantly, the study extends previous work by a) using fear-relevant stimuli that are more common objects of fears in pathological fear in anxiety disorders; b) using fear potentiated startle (FPS) as a measurement of fear, that is thought to reflect the affective component of fear; and c) demonstrating a context-independent effect of the vicarious extinction procedure. These findings underscore reconsolidation and the interference of the process using a behavioral manipulation as a promising approach to dampen the expression of fear memory, shedding new light to the ongoing debate. Given that it is often not possible to administer an intervention or pharmacological agent at an initial trauma or triggering event, the possibility of later manipulating fear memory by disrupting reconsolidation is of particular clinical relevance. By providing a safe, non-invasive and easily implemented behavioral intervention that captures these adaptive purposes of reconsolidation, this study can contribute to finding a long-term cure for patients suffering from anxiety disorders.
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Appendix 1: Description and Trial-by –trial graph of the whole sample.
The whole sample consisted of 32 undergraduate students (10 males; age M = 20.62 years, SD = 1.8 years, range = 18-26 years).
Table 3
Mean values (SD) of age, trait anxiety, reported spider fear, level of the unconditioned stimulus (mA) and perceived intensity of the US (n=32).
* Levene’s Test for Equality of Variances was significant (F = 8.59, p = .00), indicating unequal variances. Therefore, degrees of freedom were adjusted for calculating the p-value (df = 29,65).
Reminded Spider (n=18) Reminded Gun (n=14) t-value (p-value); df = 32
Age 20.78 (2.01) 20.33 (1.70) 0.75 (.451) STAI Trait 31.78 (6.41) 35.56 (6.96) -1.63 (.120) FSQ 28.78 (13.90) 33.25 (10.02) -1.04 (.298) US Level 36.67 (26.05) 33.28 (24.79) 0.392 (.690) US Intensity Day 1 2.56 (0.71) 3.00 (0.53) -1.27 (.240)* US Intensity Day 3 2.50 (0.52) 2.75 (0.86) -1.25 (.357)
Figure 4. Trial-by-trial mean fear-potentiated startle (FPS) responses of the whole sample (n=32) elicited during the presentation of each stimulus (CS+r, CS+nr and CS-), and during the noise lone trials (NA) across the three stages: acquisition (A1-A5), vicarious extinction (E1-E10), and re-extinction (R1-R5). Error bars represent the standard error of the mean (SEM). The Y-axis is scaled to T-scores. Note that the critical analysis of the end of vicarious extinction (Day 2) to the first block of re-extinction (Day 3) revealed the same pattern as that of the subsample. That is, only a significant increase in FPS emerged to the CS+nr (: t(31)= -2.36, p = 0.025) but not to the CS+r and the CS- (both t’s < 2).
40 45 50 55 60 65 70
A1 A2 A3 A4 A5 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 R1 R2 R3 R4 R5
Fe ar P ot en ti at ed S ta rt le ( t-sc or e) CS+reminded CS+ non-reminded CS- ITI Reminder
Appendix 2: Psychometric Characteristics Questionnaires
Fear of Spiders Questionnaire (FSQ; Szymanski & O’Donohue (1995)). Participants’ self-reported fear of spiders will be assessed with the Dutch version of the FSQ (Muris & Merckelbach, 1996). The 18 items are rated on an 8-point Likert scale, ranging from 0 ‘completely disagree’ to 7 ‘completely agree’. The Dutch FSQ has good psychometric properties and is more sensitive in measuring fear in the non-clinical range than the Spider Phobia Questionnaire (SPQ) (Muris & Merckelbach, 1996).
Trait Anxiety Inventory (STAI-T; Spielberger et al., 1970). Trait Anxiety will be assessed with the Dutch Version of the STAI-T: the Zelf-Beoordelings Vragenlijst (ZBV). Although the questionnaire consists of two separate parts, only the STAI-trait will be used, as it is a three-day paradigm. All items are rated on a 4-point scale (e.g., from “Almost Never” to “Almost Always”). Both test-retest reliability and internal consistency for the trait anxiety scale is high. Moreover, good construct and concurrent validity of the scale is confirmed (van der Ploeg, Defares & Spielberger, 1980).
The Interpersonal Reactivity Index (IRI; Davis, 1980). Participants’ self reported empathic tendencies will be assessed with the Dutch version of the IRI. The questionnaire has satisfactory internal consistency, good scale reliability, satisfactory construct - (as reflected in scale inter-correlations and gender differences), discriminant - and convergent validity (de Corte, Buysse, Verhoefstadt, Roeyers, Ponnet & Davis, 2007).
